新的长链非编码RNA Lnc5q21.2在结肠癌中的作用机制研究

Colorectal cancer is one of the most common malignancies in China, and the incidence is increasing. The function and the mechanism of lncRNAs in colorectal cancer remains unclear. A group of lncRNAs related to colorectal cancer was identified in our previous study. The expression of Lnc5q21.2 is increased in human colorectal cancer. Lnc5q21.2 promotes colony formation and cell proliferation. This study will further investigate the function and the mechanism of Lnc5q21.2 on colorectal carcinogenesis. 1) The function of Lnc5q21.2 on tumor growth will be evaluated by Lnc5q21.2 expressed and unexpressed colorectal cancer cell xenograft mice model. 2) Proteins interacted with Lnc5q21.2 will be isolated and identified by ChIRP, CLIP, mass spectrometry and western blot assay. 3)The interaction of Lnc5q21.2、MYH9 and histone modification, as well as DNA methylation related proteins will be explored to understand the mechanism of Lnc5q21.2 in regulation of targeting gene expression. The function of MYH9 in human colorectal carcinogenesis will be investigated. 4) The interaction of Lnc5q21.2 and DNMTs on the regulation of gene expression will be studied by knocking out or knocking down Lnc5q21.2 with CRISPR/Cas9 system and shRNA in HCT116 and DKO cells（DNMT1、DNMT3b knock out HCT116 cells）. 5) The interaction of Lnc5q21.2 and DNMTs or histone modification related enzymes, and the structure will be predicted by computer simulation. Under the supporting of the project, we will find a group of lncRNA markers for the early detection, prognosis and chemo-sensitivity in colorectal cancer.

前期研究应用高通量测序等技术分离出一批候选的与结肠癌相关的lncRNAs，并鉴定和克隆一组全长lncRNAs；其中Lnc5q21.2在结肠癌中高表达。本课题拟1）研究Lnc5q21.2在结肠癌细胞中的功能和对裸鼠体内致瘤能力的影响；2）应用ChIRP、CLIP、质谱等技术分离和鉴定与Lnc5q21.2结合的蛋白；3）分析Lnc5q21.2、MYH9和组蛋白修饰相关蛋白的相互作用及对下游基因的表达调控机制。4）应用shRNA敲降和CRISPR/Cas9基因敲除技术研究Lnc5q21.2和DNMTs的相互作用及对靶基因表达的调控；5）分析Lnc5q21.2和DNA甲基化酶、组蛋白修饰相关酶的相互作用，以及Lnc5q21.2与其它生物大分子相互作用的结构。可望获得用于结肠癌早期诊断、预后和化疗敏感性的lncRNAs标志物；明确Lnc5q21.2在结肠癌发生过程中表观遗传调控机制。

在本课题资助下，我们发现一个新的基因间的长链非编码RNA Lnc5q21.2并明确其基本功能； Lnc5q21.2主要定位于细胞核内，Lnc5q21.2与HOXA10相互作用，并明确其结合的结构域；Lnc5q21.2通过与HOXA10直接结合抑制EMX2的表达，从而激活Wnt信号通路，在体内外促进结肠癌发生发展。进一步的研究发现Lnc5q21.2通过激活Wnt信号通路而参与DNA的损伤修复，Lnc5q21.2高表达增加结肠癌细胞对ATR抑制剂和顺铂的敏感性，与ATR抑制剂具有“协同致死”作用。Lnc5q21.2是结肠癌早期诊断、预后和化疗敏感性的新的标志物，并可作为ATR抑制剂“协同致死”治疗的靶标。在该课题的资助下我们还在食管癌中鉴定出长链非编码RNALncR39，其在68%食管癌组织高表达。LncR39能够促进食管癌的增殖和侵袭迁移、抑制细胞的凋亡，在体内外促进食管癌细胞生长。DNA损伤修复基因RASSF1A在食管癌中的甲基化率为11.57%（121/1046），在食管不典型增生中的甲基化率为1.71%（3/175），RASSF1A甲基化沉默是ATR抑制剂的敏感性标志物。在803例食管癌中发现CHFR、MGMT、MLH1、SLFN11的甲基化率分别为64.59%、12.81%、3.52%和30.25%，并研究了这些基因甲基化在“协同致死”治疗中的价值，获得了基于这些基因甲基化的“协同致死”新策略。另外，在结肠癌中研究了RAI2和ZNF331的表达调控机制和功能；在肝癌中研究了HOXD10的功能和机制；在食管癌中分析了DNA m6A甲基化的水平。